Question 12.38

A conditional mutation expresses its mutant phenotype only under certain conditions (the restrictive conditions) and expresses the normal phenotype under other conditions (the permissive conditions). One type of conditional mutation is a temperature-sensitive mutation, which expresses the mutant phenotype only at certain temperatures.

Strains of E. coli have been isolated that contain temperature-sensitive mutations in the genes encoding different components of the replication machinery. In each of these strains, the protein produced by the mutated gene is nonfunctional under the restrictive conditions. These strains are grown under permissive conditions and then abruptly switched to the restrictive condition. After one round of replication under the restrictive condition, the DNA from each strain is isolated and analyzed. What characteristics would you expect to see in the DNA isolated from each strain with a temperature-sensitive mutation in its gene that encodes in the following proteins?

• a. DNA ligase
• b. DNA polymerase I
• c. DNA polymerase III
• d. Primase
• e. Initiator protein

Question 12.39

DNA topoisomerases play important roles in DNA replication and in supercoiling (see Chapter 11). These enzymes are also the targets for certain anticancer drugs. Eric Nelson and his colleagues studied m-AMSA, one of the anticancer compounds that acts on topisomerase enzymes (E. M. Nelson, K. M. Tewey, and L. F. Liu. 1984. Proceedings of the National Academy of Sciences 81:1361–1365). They found that m-AMSA stabilizes an intermediate produced in the course of the topoisomerase’s action. The intermediate consisted of the topoisomerase bound to the broken ends of the DNA. Breaks in DNA that are produced by anticancer compounds such as m-AMSA inhibit the replication of the cellular DNA and thus stop cancer cells from proliferating. Propose a mechanism for how m-AMSA and other anticancer agents that target topoisomerase enzymes taking part in replication might lead to DNA breaks and chromosome rearrangments.

Question 12.40

The regulation of replication is essential to genomic stability, and, normally, the DNA is replicated just once every eukaryotic cell cycle (in the S phase). Normal cells produce protein A, which increases in concentration in the S phase. In cells that have a mutated copy of the gene for protein A, the protein is not functional and replication takes place continuously throughout the cell cycle, with the result that cells may have 50 times the normal amount of DNA. Protein B is normally present in G₁ but disappears from the cell nucleus in the S phase. In cells with a mutated copy of the gene for protein A, the levels of protein B fail to disappear in the S phase and, instead, remain high throughout the cell cycle. When the gene for protein B is mutated, no replication takes place.

Propose a mechanism for how protein A and protein B might normally regulate replication so that each cell gets the proper amount of DNA. Explain how mutation of these genes produces the effects just described.